Phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11;PFK) is a key regulatory enzyme of glycolysis. The human PFK isozymes (HPFK) are encoded by the muscle (M), liver (L) and platelet (P) genes which are differentially expressed during development and display distinct tissue specificities. HPFK-M is the predominant subunit in skeletal muscle, heart, and brain where metabolic energy requirements are principally met by glycolysis. The recessively inherited deficiency for the HPFK-M subunit, Tarui's disease (glycogen storage disease type VII [GSD type VII]) is most commonly characterized by exertional myopathy and compensated hemolysis. In patients with this disease, PFK activity is essentially absent from muscle and approximately 50% of the normal level in red blood cells suggesting that the lack of HPFK-M activity is responsible for the observed clinical symptoms. The long term objectives of this study have been to determine the molecular basis of the defect(s) in the HPFK-M gene responsible for GSD type VII and to understand the regulation of the developmental and tissue-specific expression of the HPFK-M gene. The isolation and characterization of HPFK-M cDNA and genomic clones has revealed the sequence of the enzymatically active HPFK-M polypeptide, the existence of an alternatively spliced HPFK-M transcript encoding an HPFK-M related polypeptide, and the existence of two promoters governing the expression of the HPFK-M gene. Therefore, a major goal of this analysis is to identify the nucleotide differences between the normal HPFK-M allele and the mutant HPFK-M alleles and to determine the effect of these changes on the enzymatic activity of the HPFK-M polypeptide (i.e. determine functionally critical amino acid residues). This will be achieved by expressing the product of the mutant HPFK-M alleles in mouse fibroblasts and characterizing its enzymatic activity. Similarly, the properties of the HPFK-M related polypeptide encoded by the alternatively spliced HPFK-M transcript will be characterized after expression in mouse fibroblasts. This analysis may indicate a role for this HPFK-M related polypeptide in the regulation of the key step in glycolysis catalyzed by PFK. The characterization of the transcriptional activity from the two HPFK-M promoters in transient transfection assays and transgenic mice should indicate the developmental and tissue-specific programme of expression governed by these regulatory elements. The transcription factors which mediate this programmed expression by binding to the regulatory elements of the two HPFK-M promoters will be examined by gel retardation and DNase 1 protection assays.